Summary

The proper development of tissues requires morphogen activity that dictates
the appropriate growth and differentiation of each cell according to its
position within a developing field. Elimination of underperforming cells that
are less efficient in receiving/transducing the morphogenetic signal is
thought to provide a general fail-safe mechanism to avoid developmental
misspecification. In the developing Drosophila wing, the morphogen
Dpp provides cells with growth and survival cues. Much of the regulation of
transcriptional output by Dpp is mediated through repression of the
transcriptional repressor Brinker (Brk), and thus through the activation of
target genes. Mutant cells impaired for Dpp reception or transduction are lost
from the wing epithelium. At the molecular level, reduced Dpp signaling
results in Brk upregulation that triggers apoptosis through activation of the
JNK pathway. Here we show that the transcriptional co-regulator dNAB is a Dpp
target in the developing wing that interacts with Brk to eliminate cells with
reduced Dpp signaling through the JNK pathway. We further show that both dNAB
and Brk are required for cell elimination induced by differential dMyc
expression, a process that depends on reduced Dpp transduction in outcompeted
cells. We propose a novel mechanism whereby the morphogen Dpp regulates the
responsiveness to its own survival signal by inversely controlling the
expression of a repressor, Brk, and its co-repressor, dNAB.

INTRODUCTION

A fundamental question in development is how growth, cell fate
specification and pattern formation are spatially and temporally coordinated
to control the final shape, size and cellular make-up of an organ. Part of the
answer resides in the ability of a single morphogenetic molecule to provide
simultaneous guiding cues for different developmental processes
(Serrano and O'Farrell, 1997).
One such signaling molecule is Decapentaplegic (Dpp), a member of the
TGFβ superfamily that provides cells of the developing
Drosophila wing with patterning, growth and survival cues.

The brinker (brk) gene is a key target of the Dpp pathway
that is negatively regulated by Dpp signaling throughout embryonic and larval
development. brk encodes a transcriptional repressor. Loss of Brk
function leads to ectopic expression of Dpp target genes, tissue overgrowth
and cell fate transformations corresponding to elevated levels of Dpp
signaling (Campbell and Tomlinson,
1999; Jazwinska et al.,
1999; Minami et al.,
1999). Moreover, removal of Brk leads to overgrowth and ectopic
expression of Dpp targets even in the absence of Dpp or other essential
components of the pathway, such as Tkv or Mad
(Jazwinska et al., 1999;
Marty et al., 2000),
indicating that to a large extent Dpp signaling acts through negative
regulation of brk expression. Interestingly, although a Dpp gradient
controls differential gene expression by gradually downregulating
brk, the slope of the Dpp gradient is not itself important for
proliferation (Muller et al.,
2003; Schwank et al.,
2008). Brk is a sequence-specific transcriptional repressor that
alternatively requires the co-repressors Groucho (Gro) and CtBP for repressing
some Dpp-responsive genes, but not for others
(Hasson et al., 2001;
Zhang et al., 2001).

In Drosophila imaginal discs, a phenomenon of cell competition has
been described in which normal cells overproliferate at the expense of
neighboring Minute cells that have reduced ribosomal protein gene dose,
eliminating them via apoptosis from developing tissues
(Morata and Ripoll, 1975;
Simpson and Morata, 1981).
Similar competitive interactions occur when cells that express more dMyc
[Diminutive (Dm) - FlyBase] or cells that are mutant for components of the
Hippo/Warts pathway, behave as super-competitors that both outgrow adjacent
wild-type cells and induce their death (de
la Cova et al., 2004; Moreno
and Basler, 2004; Tyler et
al., 2007). Competition for the Dpp survival signal appears to be
the driving force behind cell competition. This notion is based on the finding
that outcompeted cells exhibit reduced Dpp signaling
(Moreno and Basler, 2004;
Moreno et al., 2002;
Tyler et al., 2007) and their
elimination can be prevented by forced expression of either Dpp or its
activated receptors (Moreno and Basler,
2004; Moreno et al.,
2002). At the molecular level, reduced Dpp signaling activity
results in failure to repress the expression of Brk, the upregulation of which
triggers apoptosis through activation of the JNK pathway.

Elimination of underperforming cells from a developing field may be a
general feature of morphogen gradients that circumvents misspecification and
the accumulation of detrimental developmental mistakes that would otherwise
lead to embryonic malformation. Here, we find that the transcriptional
co-regulator dNAB (Nab - FlyBase) is a target of Dpp in the wing primordium
that interacts with Brk to promote JNK-mediated elimination of cells with
impaired Dpp signaling. We further demonstrate that both dNAB and Brk are
required for dMyc-induced cell competition. In contrast to Gro, a known
co-repressor of Brk, dNAB is not required for Dpp-dependent patterning,
whereas Gro does not promote JNK-mediated cell death.

MATERIALS AND METHODS

Fly strains and transgenes

We used the following fly strains: brkM68,
dnabR6H8, dnab-lacZSH143, omb-lacZ, vg-lacZ,
puc-lacZE69, arm-lacZ, brk-lacZX47,
UAS-tkvQ235D, UAS-gro, UAS-Dad, UAS-brk,
UAS-puc and EP-dnab. P-element S149, which maps 23 bp
upstream of the 5′ end of the dnab transcription unit, was
replaced with the y+-marked EP element DA530inv
(Gerlitz and Basler, 2002)
essentially as described by Sepp and Auld
(Sepp and Auld, 1999).
Transgenes were expressed using the Gal4/UAS binary system with the following
drivers: hh-Gal4, sd-Gal4 and C765-Gal4.

For MARCM experiments, genotypes of dissected larvae were as follows:
sd-Gal4/hs-flp; UAS-Dad/UAS-GFP; tubP-Gal80 FRT80/dnabR6H8
FRT80 and sd-Gal4/hs-flp; UAS-Dad/UAS-GFP; tubP-Gal80
FRT80/FRT80. Clones were induced by heat shock for 60 minutes at
37°C. Larvae were dissected at 48, 72 and 96 hours after clone
induction.

Wild-type clones in a tub>dmyc background

Larvae of genotype yw hsp70-flp; tub>dmyc>Gal4; UAS-GFP with
or without UAS-RNAi constructs
(Dietzl et al., 2007) to knock
down dnab (#6273) or brk (#2919), were heat shocked for 15
minutes at 37°C and dissected after 24, 48 or 72 hours. UAS-RNAi
constructs against other Drosophila genes (more than 50 different
genes) were also used and most of them (>90%) did not cause the rescue that
was observed when knocking down either brk or dnab.

Plasmid construction

Molecular manipulations were conducted according to standard protocols.
Constructs containing full-length dnab cDNA and its derivatives were
prepared by standard PCR amplification. Following sequencing, these were
inserted in-frame into the pGEX-2T vector. A PCR-amplified full-length
brk was cloned into the pET17b vector.

Cleaning of the R6H8 chromosome and rescue experiment

In order to clean the chromosomal region distal and proximal to the
dnabR6H8 mutation
(Nairz et al., 2004), two
y+-marked P-insertions located in close proximity to the
dnab gene (W158 at 63B and W55 at 65D, our
unpublished results) were sequentially first recombined on to the
dnabR6H8 mutant chromosome and then removed by
recombination.

Two independent P[genomic dnab] insertions on the third chromosome
rescued the lethality of homozygous dnab mutant flies (S149 and
dnabR6H8). Flies carrying the P[genomic dnab] and
the dnab mutations on the third chromosome lost the Tm6B balancer
chromosome.

RNA in situ hybridizations

RNA in situ hybridizations were carried out according to standard
protocols. DIG (Roche) RNA probes were synthesized from a template derived by
PCR from genomic DNA using the following primers: dNAB_fw,
5′-AGACCATCTGGCTGCTGACC-3′ and dNAB_rev,
5′-AATTAACCCTCACTAAAGGTCTGGTGAAGCAGCACTCC-3′.

GST pull-down experiments

GST pull-down experiments were carried out according to standard protocols,
essentially as described by Hasson et al.
(Hasson et al., 2001).

RESULTS

dnab is a Dpp target in the wing disc

The Dpp signaling pathway controls growth, survival and patterning during
Drosophila appendage development. To identify novel putative Dpp
targets, we screened a collection of 2000 wing disc Gal4 enhancer-trap
insertion lines (Gerlitz et al.,
2002) with a UAS-GFP reporter for lines that exhibit
expression patterns centered on the stripe of dpp expression at the
anterior-posterior compartment boundary. The responsiveness of each of the
selected enhancer-trap lines to changes in Dpp signaling was then assessed
using UAS transgenic lines of either a constitutively active form of the
type-I Dpp receptor thickveins (tkvQ235D)
(Nellen et al., 1996), or of
Daughters against dpp (Dad)
(Tsuneizumi et al., 1997), an
inhibitor of the Dpp pathway. Using this selection process, we identified
S149, a lethal Gal4 insertion, the expression of which is confined to the
presumptive wing blade (Fig.
1A). Through plasmid rescue of the mutagenic P-element and RNA in
situ hybridization (Fig. 1B),
we identified this insertion as a hypomorphic mutant allele of Drosophila
nab (dnab) (Clements et al.,
2003), a member of the NAB family of transcriptional co-repressors
(Russo et al., 1995;
Svaren et al., 1996).
Complementation crosses with a known lethal deletion allele
(dnabR6H8) (Nairz et
al., 2004) and introduction of a 9.5 kb genomic rescue construct
of dnab confirmed that the observed lethality is due to disruption of
dnab. We further found that the expression of a dnab-lacZ
reporter gene (SH143) (Oh et al.,
2003) is ectopically induced by misexpression of
TkvQ235D and is abolished by overexpression of either Dad
or brk (Fig. 1E-J),
establishing that dnab expression is positively regulated by Dpp
signaling in the developing wing. We have no indication as to whether this
regulation is direct or indirect. Interestingly, the reduction of
dnab-lacZ expression resulting from Brk overexpression was not as
robust as that resulting from Dad overexpression (compare Fig.
1G,H with
1I,J), suggesting that
dnab expression is regulated by Dpp/Mad signaling and not only
through the removal of brk repression. It is important to note that,
as with other wing-specific genes, dnab expression has been shown to
depend on Vestigial (Vg) (Terriente Felix
et al., 2007). Since vg is a Dpp target, it is possible
that Dpp regulates dnab expression at least in part through
regulation of vg. The fact that not every
TkvQ235D-expressing clone was able to induce dnab
expression (Fig. 1E) might
indicate that dnab expression requires inputs from both Vg and Dpp
signaling, as is the case for other Dpp target genes such as spalt
(sal; salm - FlyBase)
(Halder et al., 1998).

dnab is a Dpp target gene expressed in the Drosophila
wing pouch. (A) Wing imaginal disc (anterior to the left, dorsal
up, in all figures) showing the activity of the S149 Gal4 enhancer-trap
insertion as revealed by UAS-GFP (green). (B) In situ
hybridization with an RNA probe derived from the dnab ORF.
(C,D) dNAB and Brk expression pattern in second and third instar
larvae. Double staining of wing discs with antibodies directed against Brk
(red) and dNAB (green) shows no overlap in second instar (C) and a slight
overlap in third instar (D) larvae. (E-J) Dpp signaling positively
regulates dnab expression. (E,F) dnab-lacZ (red) is
ectopically expressed in clones overexpressing TkvQ235D (green,
arrowheads); in all figures, overexpression clones (marked with GFP, green)
are derived from activation of a Flp-able actin promoter (unless stated
otherwise). Dad (G,H) and Brk (I,J) overexpression (using the hh-Gal4
driver, green) downregulates the expression of the dnab-lacZ reporter
(red).

dNAB is not required for Dpp-dependent patterning

Motivated by the idea that the global patterning and growth activities of
the Dpp pathway are executed by its downstream targets, we first analyzed the
effect of dNAB overexpression on wing development. We found that dNAB
overexpression in the wing disc represses the expression of the Dpp/Brk target
genes sal and vg (Fig.
2A-F). To analyze the role of dNAB in wing development, we
generated homozygous clones mutant for dnab in heterozygous wing
discs. However, we found that loss of dnab function affects neither
the size of the clones, which were similar to their sibling twin spots, nor
the expression of known Dpp target genes in the wing disc, such as
optomotor-blind (omb; bifid - FlyBase),
sal and vg (Fig.
2G-O), indicating that dNAB function is not required for
Dpp-dependent growth and patterning in this tissue.

dNAB induces JNK-mediated cell death

Dpp is essential for cell survival in the wing disc, and mutant cells
impaired for Dpp reception or transduction are lost from the wing epithelium
(Adachi-Yamada and O'Connor,
2002; Burke and Basler,
1996; Moreno et al.,
2002). To test the possibility that dNAB is involved in processes
that regulate cell viability, we generated cell clones overexpressing dNAB in
a wild-type background and followed their descendents at various time points
after induction. We found that these cells undergo apoptosis, as shown by the
dramatic increase in the levels of activated Caspase 3, and that they are
gradually eliminated first from the periphery of the wing disc where
brk levels are high, and subsequently from the medial region
(Fig. 3A-D).

The JNK pathway mediates apoptosis in various developmental contexts
(Adachi-Yamada et al., 1999;
McEwen and Peifer, 2005),
including the elimination of cells with impaired Dpp signaling
(Adachi-Yamada et al., 1999;
Adachi-Yamada and O'Connor,
2002; Moreno et al.,
2002). We asked whether JNK activation is involved in dNAB-induced
cell death. The extent of activation of the JNK pathway can be monitored
through the expression of the target gene puckered (puc),
which encodes a protein phosphatase that negatively regulates the pathway
(Martin-Blanco et al., 1998).
We found that overexpression of dNAB induces the expression of puc
(Fig. 3E,F), indicating that
JNK signaling is activated in the dying cells. Furthermore, when JNK signaling
was experimentally downregulated in clones of cells overexpressing dNAB by
co-expression of puc, Caspase 3 activation was to a large extent
inhibited (Fig. 3G,H), and the
clones were distributed randomly throughout the wing disc. These results
demonstrate that dNAB induces cell death through induction of the JNK pathway,
which in turn triggers Caspase-3-mediated apoptosis.

dNAB-induced apoptosis is Brk-dependent

In order to elucidate whether dNAB promotes cell death through the Dpp
signaling pathway, dNAB overexpression was combined with Brk loss-of-function,
a dedicated downstream effector of the Dpp pathway. To this end, clones mutant
for brk were generated in the posterior compartment where dNAB was
overexpressed using a hedgehog (hh)-Gal4 driver
(Fig. 4A-C). We found that
dNAB-induced apoptosis is completely nullified by loss of Brk function, as
evidenced by the reduction of activated Caspase 3 to normal levels
(Fig. 4B). Thus, the cell
death-promoting activity of dNAB is Brk-dependent, and therefore functions
through the Dpp signaling pathway.

dNAB overexpression represses Dpp target genes, but is not required for
Dpp-dependent growth and patterning in the Drosophila wing disc.
(A-C) dNAB-overexpressing clone (A, green) represses Sal expression as
revealed by immunostaining (B, red). (C) Merge of A and B. (D-F)
hh-Gal4 driver was used to overexpress dnab in the
posterior compartment (D, green). vg expression, as visualized by
means of the vg-lacZ reporter, is abolished by dnab
overexpression (E, red). (F) Merge of D and E. (G-O) In dNAB
loss-of-function clones (G,J,M), marked by the loss of GFP (arrowheads), the
expression of the Dpp target genes omb (H, red), sal (K,
red) and vg (N, red) is unaffected. (I,L,O) Merges.

Epistatic positioning of dNAB within the Dpp signaling pathway

To determine the epistatic position of dNAB within the Dpp pathway, we
co-expressed dNAB with TkvQ235D. We found that excessive activation
of the Dpp signal transduction pathway by TkvQ235D does not rescue
dNAB-induced apoptosis (Fig.
4G-I). Furthermore, expression of TkvQ235D is known to
produce abnormally large discs, whereas co-expression of dNAB with
TkvQ235D partially reversed this effect of overgrowth such that
wing discs varied in size, ranging from small through normal to large
(Fig. 4D-F; data not shown).
Thus, dNAB appears to affect cell survival by impinging on the Dpp signaling
pathway downstream of the receptor complex.

We next tested whether dNAB acts through transcriptional repression of
brk (Marty et al.,
2000; Muller et al.,
2003) at the level of Mad, similar to the mode of action of the
inhibitory Smad, Dad (Tsuneizumi et al.,
1997). According to this possibility, excess dNAB should interfere
at the level of brk transcriptional repression and lead to its
accumulation. On the contrary, we found that dNAB overexpression, while
inducing apoptosis, had no effect on the expression levels of a
brk-lacZ reporter (Fig.
4J-L). Thus, dNAB acts downstream of brk transcriptional
regulation.

dNAB and Brk physically interact in vitro

The fact that overexpression of dNAB represses Dpp/Brk target genes and
that dNAB has been shown to act as a transcriptional co-regulator in
Drosophila (Terriente Felix et
al., 2007; Tsuji et al.,
2008) prompted us to assess the possibility that dNAB physically
interacts with Brk. Using a GST pull-down assay, we found that like Gro, a
known Brk co-repressor, dNAB binds directly to the Brk protein
(Fig. 5A). We then used
sequential fragments of the dNAB protein to narrow down the Brk-binding region
of dNAB to the NAB conserved domain 2 (NCD2)
(Fig. 5B,C), a region found in
the C-terminal half of all NAB proteins that contains a bipartite-like nuclear
localization sequence and the transcriptional repression function
(Swirnoff et al., 1998). Taken
together, our results demonstrate that dNAB acts together with the Brk
repressor, apparently through direct protein-protein interactions.

dNAB promotes cell elimination induced by impaired Dpp signaling

It is well documented that cells impaired for Dpp signaling, due to removal
of the Dpp receptor or to forced expression of the Dpp pathway inhibitor Dad,
or of Brk, are first eliminated from the center of the wing disc and
subsequently from lateral regions, where normally brk is expressed
and dnab is not (Adachi-Yamada and
O'Connor, 2002; Burke and
Basler, 1996; Moreno et al.,
2002). Thus, dNAB is not essential for cell elimination induced by
very high levels of Brk. We investigated whether loss of dNAB function affects
cell removal from the wing pouch region induced by reduced Dpp signaling.
Using the MARCM system (Lee and Luo,
2001) in combination with a wing Gal4 driver [scalloped
(sd)-Gal4], we generated dnab loss-of-function
clones that overexpressed the Dpp pathway inhibitor Dad in the wing disc. We
favored this experimental set-up because activation of the UAS transgene is
dependent on Gal80 perdurance, and therefore should allow dNAB protein to
dissipate in the loss-of-function clones prior to Dad accumulation and the
subsequent upregulation of brk. Seventy-two hours after clone
induction, we found a greater than 2-fold increase (two-tailed test,
P<0.005) in the number of dnab loss-of-function clones
that survived in the wing pouch region compared with control clones
(Fig. 6A-E). In addition, in
many cases we observed higher levels of active Caspase 3 in control clones as
compared with dnab loss-of-function clones
(Fig. 6B,D). We conclude that
dNAB promotes the elimination of cells with reduced Dpp signaling.

The fact that dNAB expression is regulated by Dpp/Brk signaling raises the
question of how clones impaired for Dpp signaling, such as Dad-overexpressing
clones, die in a dNAB-dependent manner, for one might expect that in such
clones dnab expression would be lost when brk expression is
gained. The simplest explanation is that the dNAB protein is stable and has a
high perdurance, so that under conditions in which Brk expression is gained
there is enough dNAB protein to allow for interaction. Consistently, we found
that dNAB protein was present in Brk-overexpressing clones that survived in
the wing pouch region 30 hours after induction
(Fig. 6I,J). Significantly, the
expression of the Dpp/Brk target gene sal was completely lost in such
clones (Fig. 6K,L), indicating
that Brk is upregulated and active when dNAB is still present.

dNAB sensitizes cells to the cell death-promoting activity of
Brk

We tested whether dNAB enhances the cell death-promoting activity of Brk.
Expression of Brk in the wing disc using a mild ubiquitous Gal4 driver (C765)
resulted in low Caspase 3 activation mainly in the wing pouch region
(Fig. 6F) where dnab
is normally expressed, whereas driving dNAB expression with the same Gal4
driver resulted in an almost complementary pattern of enhanced Caspase 3
activation in the wing periphery (Fig.
6G). Importantly, co-expression of dNAB and Brk resulted in
significantly smaller wing discs and a dramatic increase in Caspase 3
activation in the wing pouch (Fig.
6H). Thus, in the presence of dNAB, lower levels of Brk induce
cell death, implying that dNAB expression in the wing pouch region sensitizes
cells to the killing activity of Brk. Altogether, these results indicate that
dNAB acts as a co-repressor that interacts with Brk to promote apoptotic
elimination of cells with reduced Dpp signaling.

dNAB and Brk are both required for dMyc-induced cell competition

Recent studies, in which apposing cell populations with different levels of
dMyc or of a Minute gene product were generated in the wing disc,
established Dpp as a crucial survival factor for which cells continuously
compete to prevent apoptosis (Moreno and
Basler, 2004; Moreno et al.,
2002). Reduced Dpp signaling activity in outcompeted cells results
in the upregulation of Brk, which in turn triggers apoptosis through
activation of the JNK pathway. The results presented so far prompted us to
investigate whether dNAB could also play a positive role in cell elimination
driven by different levels of dMyc. Using the transgene
tub>dmyc>Gal4, we generated wild-type cells surrounded by cells
expressing extra dMyc and found, in accordance with previous results
(de la Cova et al., 2004;
Moreno and Basler, 2004), that
they were rapidly lost from the wing primordium
(Fig. 6M; see Fig. S1 in the
supplementary material). However, knocking down the expression of either
dnab or brk specifically in the wild-type cells [using the
appropriate UAS-RNAis (Dietzl et
al., 2007)] led, remarkably, to their rescue and reversed their
proliferation deficit (Fig.
6N-P). Notably, dnab RNAi appeared to result in
ragged-edge clones, whereas brk knockdown led to round clones,
indicating that unlike Brk, dNAB has no apparent role in cell affinity. From
these results, we concluded that both dNAB and Brk play a crucial role in
mediating dMyc-induced apoptotic cell competition.

dNAB and Gro qualitatively differ in their ability to induce
JNK-mediated cell killing

The results presented above raised the possibility that the previously
identified co-repressor of Brk, Gro
(Hasson et al., 2001;
Zhang et al., 2001), which has
been implicated in patterning, could play a role similar to that of dNAB in
promoting Brk-dependent cell elimination. However, several lines of evidence
appear to contradict this idea. First, in contrast to the situation in which
overexpression of dNAB leads to rapid cell loss
(Fig. 3A-D), clones of cells
overexpressing Gro appear large in size, do not show Caspase 3 activation and
are not readily eliminated, but rather are distributed randomly throughout the
wing disc, including the lateral regions where brk is highly
expressed (Fig. 7A-C). Notably,
these Gro clones readily repress the Brk target genes sal, omb and
vg (Hasson et al.,
2001; Zhang et al.,
2001). Second, whereas excess dNAB leads to induction of both JNK
signaling and Caspase 3 activation (Fig.
7D-F), overexpression of Gro throughout the entire posterior
compartment shows neither of these effects
(Fig. 7G-I). Thus, dNAB and Gro
qualitatively differ with respect to their ability to induce JNK-mediated cell
killing and Dpp-mediated patterning.

DISCUSSION

Morphogens are secreted signaling molecules that organize a developing
field by determining the growth and fate of responding cells according to the
level of the morphogen they perceive. Elimination of underperforming cells
with improper reduced morphogenetic signaling that would otherwise assume an
inappropriate positional identity is thought to provide a corrective mechanism
to circumvent aberrant development. Here we report on the identification of
the transcriptional co-regulator dNAB as a target and an effector of the Dpp
morphogen in the developing wing and demonstrate that dNAB is required for the
elimination of cells with impaired Dpp signaling.

NAB proteins comprise a family of transcriptional co-regulators implicated
in various developmental processes in different organisms. Drosophila
NAB was found to be required for determining specific neuronal fates in the
embryonic CNS and for wing hinge patterning
(Clements et al., 2003;
Tsuji et al., 2008). Our work
shows that dNAB induces cell elimination through induction of the JNK pathway,
which in turn triggers Caspase-3-mediated apoptosis. We show that dNAB acts as
a co-repressor that interacts with Brk to induce apoptotic cell elimination.
This conclusion is based on several lines of evidence. First, dNAB-induced
apoptosis is completely nullified by removal of Brk. Second, our epistatic
analysis placed dNAB in the Dpp signaling pathway downstream of the receptor
complex and of brk transcriptional repression and upstream of Brk.
Third, dNAB physically associates with Brk through its NCD2 domain in vitro.
Fourth, dNAB enhances the killing activity of Brk in the presumptive wing
blade region and is required for elimination of Dad-overexpressing cells, a
process that is completely dependent upon Brk function. Finally, ectopic
expression of dNAB represses the expression of Dpp/Brk target genes.

Competitive interactions occur between cells differing in their levels of
dMyc, such that cells expressing more dMyc both outgrow neighboring cells and
induce their death (de la Cova et al.,
2004; Moreno and Basler,
2004). This competitive behavior correlates with, and can be
modulated by, the activation of the Dpp survival signaling pathway, showing
that dMyc-induced cell competition relies on Dpp signaling. The fact that
dNAB, similar to Brk, is crucial for dMyc-induced cell competition strongly
supports a role for dNAB as an effector of cell elimination of underperforming
cells with reduced Dpp signaling.

Elimination of underperforming cells takes place only during early larval
stages. Clones generated later, during the third instar larval stage, persist
to adulthood (e.g. Burke and Basler,
1996; Morata and Ripoll,
1975; Simpson,
1979). Consistently, using double staining of wing discs with
antibodies directed against Brk and dNAB, we have found that the two do not
overlap in the second instar larval stage [60 hours after egg laying (AEL)]
(Fig. 1C) and only slightly
overlap during the third instar (80 hours AEL)
(Fig. 1D). These findings
suggest that the Brk-dNAB complex is active in cell elimination only during
early development. This might indicate that either another factor required for
complex activity is present only during early development, or that a factor is
present during later stages that inhibits the complex. Alternatively,
intensive growth/proliferation might be required for the execution of the
killing activity of the complex.

The morphogen Dpp acts through a well-characterized transduction pathway to
simultaneously regulate growth, survival and patterning. To a large extent,
Dpp signaling acts through negative regulation of brk expression.
This implies that a complete answer to how the Dpp signal directs different
cellular and developmental processes requires an understanding of how Brk
executes its transcriptional repression functions. Our finding that dNAB is a
Brk co-repressor is in accordance with recent results showing that
overexpression of Brk forms that cannot bind either Gro or CtBP results in
repression of sal, omb and vg, and that Brk contains
additional co-repressor-binding domains
(Winter and Campbell, 2004).
We found that in contrast to Gro, a known co-repressor of Brk, the function of
dNAB is not required for Dpp-dependent patterning. However, Gro does not play
a similar role to that of dNAB in promoting JNK-mediated cell killing. These
findings imply that the choice of Brk co-repressor determines the specificity
of target gene repression, thereby modulating different Dpp outputs.
Mechanistically, this could be achieved in a number of ways: for example, dNAB
or Gro association might alter the DNA-binding specificity of Brk, or the
promoters of Brk target genes might be differentially responsive to dNAB and
Gro. In addition, the fact that Gro is ubiquitously expressed throughout the
developing wing, and that Dpp induces dNAB expression in the center of the
wing disc while restricting Brk expression to lateral regions, provide another
means for differentially modulating Dpp outputs.

dNAB interacts with Brk to eliminate cells with reduced Dpp signaling
and is required for dmyc-induced cell competition. (A-E)
dNAB loss-of-function decreases the elimination of Dad-overexpressing clones.
(A-D) dNAB loss-of-function clones overexpressing Dad (A, green) visualized 72
hours after induction are significantly more abundant in the pouch region
compared with control clones (C, green). Activated Caspase 3 levels in control
clones (D, red) are higher than in dNAB loss-of-function clones (B, red). (E)
Bar chart showing a 2-fold increase (P<0.005) in the average
number of dNAB loss-of-function clones overexpressing Dad (left column, blue,
n=51) that survived in the wing pouch region compared with wild-type
control clones (right column, red, n=54). (F-H) dNAB enhances
the killing activity of Brk in the wing pouch region (outlined by dashed
line). The C765-Gal4 driver was used to drive ubiquitous (mild)
expression of brk and dnab in the wing disc. (F) Wing disc
overexpressing Brk shows mild Caspase 3 activation (red) in the wing pouch
region. (G) Wing disc overexpressing dNAB shows strong Caspase 3 activation
(red) at the periphery around the wing pouch. (H) Wing disc overexpressing
both Brk and dNAB shows dramatic enhancement of Caspase 3 activation (red) in
the pouch region. The delineation of the pouch is based on the natural folds
of the disc seen in bright-field microscopy. All discs were photographed under
the same magnification (×200). Discs that co-express dnab and
brk (H) are much smaller than those that express either gene alone
(F,G). (I-L) Stability of dNAB protein in brk-overexpressing
clones (generated by Flp-able abx-ubx promoter). (I,J) dNAB protein
levels in the wing pouch, as revealed by immunostaining (red), are not
affected in GFP-marked brk-overexpressing clones (arrowhead, green)
30 hours after induction. (K,L) Sal protein levels, as revealed by
immunostaining (red), are diminished in brk-overexpressing clones
(arrowhead, green) 30 hours after induction. (M-P) Brk and dNAB are
required for dMyc-induced cell competition. (M-O) GFP-marked clones of
wild-type cells (green) generated in a tub>dmyc genetic background
and monitored for survival 72 hours after induction. (M) Most clones have
disappeared from the wing pouch, and only a few persist in the periphery
(n=50). (N,O) When expressing a UAS-RNAi construct that
knocks down the expression of either brk (N) or dnab (O),
many clones persist in the wing pouch (n=50). (P) Bar chart showing
the percentage of the wing pouch area occupied by GFP-marked wild-type cells
recovered in M-O. To measure the percentage of wild-type cells that survived,
each wing pouch was subdivided into four quadrants and the surviving
GFP-marked clones were fitted into the quadrants. Discs were analyzed either
by computer imaging or by hand and were assigned to one of four groups
according to the percentage of quadrants occupied: 0-10, 25, 50 or 75%.

Based on our findings, we propose a molecular model to explain how the
morphogen Dpp regulates the cellular response to its own survival signal in
the developing wing by inversely controlling the expression of two key
factors, Brk and dNAB. In the center of the wing disc, Dpp represses
brk and induces dnab expression, so that in situations in
which Dpp signaling activity is abnormally reduced, the resulting local
increase in the levels of Brk, which complexes with dNAB, activates the
apoptotic pathway. Thus, the Dpp signal sensitizes cells in the center of the
wing disc to the apoptotic effect associated with reduced Dpp signaling by
maintaining dNAB expression. In lateral regions of the wing disc, where Brk
expression is normally higher, apoptotic cell elimination is attenuated, at
least in part owing to a lack of dNAB. Thus, by invoking dNAB as a Dpp
effector molecule that sensitizes cells to the levels of Brk, we can at least
in part explain why cells in the center of the wing disc, near the Dpp source,
are more susceptible to cell elimination induced by reduced Dpp signaling, and
why high levels of Brk in the periphery do not necessarily bring about
apoptosis.

Given that dNAB appears to play no role in Dpp-mediated patterning, we
propose that dNAB functions in the wing to prevent developmental errors and
discontinuities along the Dpp signaling gradient. This mechanism might be a
general feature of morphogen gradients that functions to avoid the
accumulation of detrimental developmental mistakes that would otherwise lead
to embryonic malformation, and is potentially important in cancer, where tumor
cells overexpressing oncogenes such as Myc may act as
super-competitors. Thus, the molecular principles underlying such
developmental fail-safe mechanisms are clearly of biomedical interest.

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